Figure 4.

Knockdown of AXL attenuates AXL-mediated signaling molecules. (A) Left, normalized mRNA expression of Gas6 in lung tissues from smokers (former and current) versus nonsmokers (never) using the GDS534 data set. Right, Gas6 mRNA expression over time in response to tobacco smoke exposure (2R4F and Lights cigarettes) using the GDS3494 data set. (B) Real-time qRT-PCR analyses of Gas6 mRNA expression in normal and smoking normal primary fibroblasts (left) and in PBS- and smoke-treated (CSE) fibroblasts (right). Data are expressed as means ± SD (n = 3). (C) Left, secreted Gas6 levels from normal and smoking normal primary fibroblasts. Right, secreted Gas6 levels from PBS- and smoke-treated fibroblasts. Data are expressed as means ± SEM; *P < 0.05 versus PBS (n = 3). (D) Left, secreted sAXL levels from normal and smoking normal primary fibroblasts. Right, secreted sAXL levels from PBS- and smoke-treated fibroblasts. Data are expressed as means ± SEM (n = 3). (E and F) Genetic knockdown of AXL to downregulate AXL levels in cells. Multiple lung fibroblast cells as indicated were treated with nonspecific (control siRNAs) or AXL-specific siRNAs (AXL siRNAs); after 72 hours of transfection, cells were subjected to Western blot analyses (E), and two biofunctional assays (F), MTS proliferation assays (left) and Matrigel transwell invasion assays (right), respectively. Data are shown as mean ± SD; *P < 0.05 versus control siRNA (n = 4). Gas6 = growth arrest–specific 6; MARCKS = myristoylated alanine-rich C kinase substrate; sAXL = soluble AXL.